Skin, mucous membranes and hair are constantly exposed to stressful factors, both of a chemical and physical nature. Solar radiation, the exposure to certain chemical agents or high temperatures can have harmful effects on the cells which make up the skin, accelerating its aging and making it look unhealthy. The mechanisms through which ultraviolet radiation (UV) exercises these effects includes the formation of reactive oxygen species, damage to the DNA, and the denaturation of proteins, among others.
Denaturation or change in the proteins' conformation can imply the exposure of hydrophobic residues at the protein surface, a situation in which the proteins are susceptible to forming aggregates, thus losing their functionality. This is dangerous for the integrity of the cell, and therefore it has specialized mechanisms to combat the aforementioned situations: all the live organisms have mechanisms to prevent the damage caused by accumulation of misfolded proteins [Ananthan J., Goldberg A. L. and Voellmy R. (1986) “Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes” Science 232:522-524].
It has been seen that the cells respond to a stressful situation by increasing synthesis of the so-called stress proteins. This response begins when the cell detects an accumulation of abnormally folded proteins, giving rise to an increase in the transcription of heat shock genes X is J. and Wu C. (1993) “Protein traffic on the heat shock promoter: parking, stalling, and trucking along” Cell 74:1-4]. The products of these genes are classified into two large groups, heat shock proteins and glucose regulated proteins. The term “heat shock protein” originates from the observation of an increase of these proteins' synthesis in cells incubated at an abnormally high temperature. These proteins' synthesis is also increased not just when the cells are subjected to an increase in temperature, but also in other stressful situations such as exposure to UV radiation, oxidative stress, osmotic shock, inflammation, hypoxia, exposure to pollutants such as heavy metals, lack of nourishment and lack of hydration [Lindquist S. (1986) “The heat-shock response” Annu. Rev. Biochem. 55:1151-1191].
Heat shock proteins are a family of proteins classified according to their molecular weight, the one that have been subject to more studies are 60 kDa and 70 kDa proteins, due to their constituent expression in all cells and their direct participation in several aspects of protein maturation. Hsp70 principally comprises two proteins: Hsp73, the form expressed constituently, and Hsp72, the inducible form, which is transcriptionally regulated by the heat shock factor protein 1 (HSF1). These proteins are also called molecular chaperones, due to their function of directing the folding of recently synthesized proteins from a globule-like conformation merged to a final compact structure, avoiding the appearance of conformations susceptible to forming aggregates and, therefore, ensuring their correct functionality. In normal conditions, Hsp70 is located in the nucleus and cytoplasm and interacts transitorily with the newborn proteins, it facilitates their folding and promotes their translocation through the Golgi complex and endoplasmic reticulum, in joint action with Hsp60. In stressful conditions, however, Hsp70 forms a complex with the unfolded proteins or erroneously folded proteins, to rescue them from degradation and irreversible damage, or the opposite, to increase the possibilities of a proteolytic attack in the event that it is impossible to protect them [Hayes S. A. and Dice J. F. (1996) “Roles of molecular chaperones in protein degradation” J. Cell. Biol. 132:255-258; Gething M. J. and Sambrook J. (1992) “Protein folding in the cell” Nature 355:33-45]. Neither Hsp70 or Hsp60 end up forming part of the final correctly folded protein, nor do they possess any specific information on the folding; they simply prevent inappropriate interactions from being established which may cause misfolding or lead to aggregations and, therefore, loss of functionality. The mechanism through which the protein adopts its definitive conformation is, however, unknown.
As well as the chaperone functions reestablishing the conformation of badly-folded proteins, the participation of Hsp70 has been described in processes of protection and repair of DNA in the case of damage caused to them by UV radiation or ionizing radiation [Bases R. (2006) “Heat shock protein 70 enhanced deoxyribonucleic acid base excision repair in human leukemic cells after ionizing radiation” Cell Stress Chaperones 11:240-249; Niu P., Liu L., Gong Z., Tan H., Wang F., Yuan J., Feng Y, Wei Q., Tanguay R. M. and Wu T. (2006) “Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner” Cell Stress & Chaperones 11:162-169].
The response to stress constitutes a universally conserved cell defense mechanism which is reflected in the so-called acquired thermotolerance, a phenomenon according to which cells that suffer a non-lethal thermal shock are capable, after a recovery period at normal growth temperature, of surviving a second thermal shock which would have been lethal the first time around [Subjeck J. R., Sciandra J. J. and Johnson R. J. (1982) “Heat shock proteins and thermotolerance; a comparison of induction kinetics” Br. J. Radiol. 55:579-584; Angelidis C. E., Lazaridis I. and Pagoulatos G. N. (1991) “Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance” Eur. J. Biochem. 199:35-39; Li G. C., Li L. G., Liu Y. K., Mak J. Y., Chen L. L. and Lee W. M. (1991) “Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene” Proc. Natl. Acad. Sci. USA 88:1681-1685]. This acquired thermotolerance has been seen to be transitory, it usually lasts between 12 and 24 hours in growing cells, and depends on the changes induced by the shock of the initial temperature, such as levels of increase in the expression and accumulation of shock proteins. Within the Hsp family it has been verified that Hsp70 is responsible for induction of thermotolerance: specific inhibition both of the transcription as well as the synthesis of Hsp72 prevents the protecting effects induced by thermal treatment [Trautinger F., Kindås-Mügge I., Barlan B., Neuner P. and Knobler R. M. (1995) “72-kD heat shock protein is a mediator of resistance to ultraviolet B light” J. Invest. Dermatol. 105:160-162; Simon M. M., Reikerstorfer A., Schwarz A., Krone C., Luger T. A., Jäättelä M. and Schwarz T. (1995) “Heat shock protein 70 overexpression affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of cytokine release” J. Clin. Invest. 95:926-33].
Subsequently it was verified that any agent or treatment capable of inducing a response to stress provides the cell with protection in the face of a subsequent exposure to a stress-causing agent, regardless of the origin of that stress [Kampinga H. H., Brunsting J. F., Stege G. J. J., Burgman P. W. J. J. and Konings A. W. T (1995) “Thermal protein denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of heat shock proteins” Exp. Cell Res. 219:536-546]. Exogenous induction of the expression of shock proteins is, therefore, a plausible strategy to prevent damage to cell proteins and, therefore, maintain cell integrity.
Described in the literature are different diseases which are caused by abnormal protein folding, such as epidermolysis bullosa [Gu L. H. and Coulombe P. A. (2005) “Defining the properties of the nonhelical tail domain in type II keratin 5: insight from a bullous disease-causing mutation” Mol Biol Cell. 16:1427-1438], which is caused by the incorrect folding of keratin caused by mutations of some amino acids in its sequence. These diseases are subject to treatment with compounds which induce an increase in the levels of heat shock proteins.
In the same way, compounds which induce an increase in the expression of heat shock proteins are used in the treatment and/or care of wounds or as adjuvants in healing and/or re-epithelialization processes. It is known that wound healing and repair processes present an increase in the expression of heat shock proteins. Specifically, induction of the expression of Hsp in the case of cutaneous trauma is specific to the location of the keratinocytes in the skin; thus, Hsp70 sees its synthesis induced in epidermis keratinocytes [Laplante A. F., Moulin V., Auger F. A., Landry J., Li H., Morrow G., Tanguay R. M. and Germain L. (1998) “Expression of heat shock proteins in mouse skin during wound healing” J. Histochem. Cytochem. 46:1291-301]. It has also been observed that the external delivery of the Hsp70 protein accelerates wound healing [Kovalchin J. T., Wang R., Wagh M. S., Azoulay J., Sanders M. and Chandawarkar R. Y. (2006) “In vivo delivery of heat shock protein 70 accelerates wound healing by up-regulating macrophage-mediated phagocytosis” Wound Repair Regen. 14:129-137]. A decrease in the quantity of Hsp70 in the skin of diabetic patients with impaired wound healing and repair has also been described [Bitar M. S., Farook T., John B. and Francis I. M. (1999) “Heat-shock protein 72/73 and impaired wound healing in diabetic and hypercortisolemic states” Surgery 125:594-601; Atalay M., Oksala N., Lappalainen J., Laaksonen D. E., Sen C. K. and Roy S. (2009) “Heat shock proteins in diabetes and wound healing” Curr. Protein Pept. Sci. 10:85-95; McMurtry A. L., Cho K., Young L. J.-T., Nelson C. F. and Greenhalgh D. G. (1999) “Expression of HSP70 in healing wounds of diabetic and nondiabetic mice” J. Surg. Res. 86:36-41]. Thus, the induction of heat shock protein synthesis of is a valid strategy for the treatment and/or care of skin and/or mucous membrane wounds and, specifically, in the healing and re-epithelialization of skin and/or mucous membrane wounds which are a consequence of diabetes.
The participation of Hsp70 in the regulation of hair growth is also known in the prior art; specifically patent application MX 2007-007622 describes the application of compounds inhibiting synthesis of Hsp70 to reduce hair growth. The implication of Hsp70 in the regulation of hair growth suggests the use of compounds capable of stimulating Hsp synthesis for the treatment and/or prevention of alopecia in order to delay hair loss or induce hair growth and, specifically, for the treatment of alopecia caused by chemotherapy as a treatment for cancer as described in patent US 2002/0001629.
Abnormal protein folding also has an effect on the skin from an aesthetic point of view. Correct elastin and collagen protein folding is fundamental to maintain the flexibility of the skin and smooth and young looking skin. Young adults' skin is particularly well adapted to respond quickly and effectively to stressful situations since it is capable of synthesizing great quantities of Hsp to protect protein folding during synthesis. However, in people of an advance age the ability to maintain correct protein folding is reduced since there is a reduction in Hsp70 synthesis with age, which causes an accumulation of damaged proteins or poorly folded and poor regulation of cell death which make the skin look old [Verbeke P, Fonager J, Clark B F, Rattan S I. (2001) “Heat shock response and ageing: mechanisms and applications” Cell Biol. Int. 25:845-857]. The effect that abnormal protein folding has on the skin from an aesthetic point of view is worsened when the skin is exposed to UV radiation, and contributes to the aspect of photoaged skin. UV radiation is capable of irreversibly damaging cells, causing cell death. However, it has been demonstrated that the exposure to high temperatures has a certain protective effect on cells, reducing the amount of cell death induced by UVB [Trautinger F., Knobler R., Hönigsmann H., M. Mayr W. and Kindås-Mügge I. (1996) “Increased expression of the 72-kD heat shock protein and reduced sunburn cell formation in human skin after local hyperthermia” J. Invest. Dermatol. 107:442-443]. This exposure to high temperatures induces Hsp synthesis. These are responsible for the photoprotective effect on the harmful effects of UV radiation observed. Thus, heat shock protein synthesis induction is a valid strategy for the treatment and/or care of the skin and/or hair with the aim of reducing, delaying and/or preventing the signs of aging and/or photoaging.
Both the cosmetic and pharmaceutical sector have carried out different tests in the development of compounds capable of stimulating heat shock protein synthesis. The role played by the heat shock proteins in different conditions, disorders and diseases is widely known in the prior art, as can found, for example, in the periodical publication Heat Shock Proteins in Biology and Medicine (Research Signpost, India) or Cell Stress and Chaperones (Springer, Netherlands), among others.
It is known that some serine protease inhibitors are capable of stimulating the production of heat shock proteins, but their high toxicity prevents their use for therapeutic purposes. Because of this, the industry needs to find agents with these properties and which can also be used risk-free for the patient's or consumer's health.
Different natural extracts which stimulate Hsp synthesis are described in the prior art, such as rye seed extracts, extracts of Opuntia ficus-indica, extracts which contain mangiferin (US 2006/0088560) or those described in documents US 2004/0228816, U.S. Pat. No. 7,128,914 or FR 2834887 among others. The difficulties of obtaining extracts with a homogenous quality and known composition and purity make their industrial development difficult, particularly in the pharmaceutical sector. Different modified synthetic peptides are also described with aldehyde or α-ketoester functions which induce Hsp synthesis, such as those described in U.S. Pat. No. 5,942,494. However, the aldehyde function is chemically incompatible with a great quantity of ingredients commonly employed in topical application formulations, also showing problems of low stability in the formulations, which limits its use in the cosmetic or dermopharmaceutical sector.
The benefit of the action of heat shock proteins on the skin, mucous membranes and/or hair could also be obtained from direct application of these proteins to the skin; mucous membranes and/or hair. In this sense, U.S. Pat. No. 5,348,945 describes the exogenous application of protein Hsp70 as a method for reducing the mortality of a tissue subjected to stressful situations and, especially, to preserve tissues which are to be used in organ transplants. The topical application of proteins with a high molecular weight presents the difficulty of their low permeability through the skin and hair, thus making its development in the cosmetic or dermopharmaceutical sector difficult.
This is why despite the great number of existing compounds and/or extracts, there is still a need to identify new compounds stimulating heat shock protein synthesis which are more effective and selective than those known in the prior art.